This invention relates to vectors suitable for expression of a desired protein in an animal cell in response to the presence of one or more steroid hormones.
Transcription of eukaryotic class II genes is regulated by a complex array of trans-acting transcription factors which bind to specific DNA sequences in the promoter of a target gene. On most promoters transcriptional preinitiation complexes are assembled at the TATA box which is generally located 25-30 base pairs (bp) 5' to the site of initiation (Buratowski et al., 56 Cell 549, 1989). Preinitiation complex assembly is regulated, in part, by factors which bind to DNA sequences known as enhancers most often situated at varying distances upstream of the TATA box/initiation site (Mitchel and Tjian, 245 Science 371, 1989; Ptashne and Gann, 346 Nature 329, 1990; and Lewin, 61 Cell 1161, 1990). Enhancers can be located immediately adjacent to the site of transcriptional initiation or several kilo-bases distant from it (Zenke et al., 5 EMBO J. 387, 1986; and Jantzen et al., 49 Cell 29, 1987). The activity of enhancer factors can be directly responsive to specific intercellular signals or indirectly via intracellular transduction pathways (Green and Chambon, 4 Trends in Genet 309, 1988; Evans, 240 Science 889, 1988; Wahli and Martinez, 5 FASEB J. 2243, 1991; Gronemeyer, 25 Ann. Rev. Genet. 89, 1991; Montiminy et al., 13 Trends in Neurolog. Sciences 184, 1990; Angel and Karin, 1072 Biochim. Biophys. Acta. 129, 1991; Lin et al., 70 Cell 777, 1991; Bauerle, 1072 Biochim. Biophys. Acta. 63, 1991; and Hunter and Karin, 70 Cell 375, 1992).
The nuclear receptors represent a family of transcriptional enhancer factors which act by binding to specific DNA sequences found in target promoters known as response elements (REs) (Green and Chambon, supra; Evans, supra; Wahli and Martinez, supra; and Gronemeyer, supra). Specific members of the nuclear receptor family represent the primary intracellular targets for small lipid soluble ligands such as steroid and thyroid hormones, retinoids and vitamin D3, and as such act as ligand-inducible transcription factors. Sequence comparisons (Krust et al., 5 EMBO J. 891, 1986) and structure-function analyses (Giguere et al., 46 Cell 645, 1986; Kumar et al., 51 Cell 941, 1987; Kumar and Chambon, 55 Cell 145, 1988; and Green and Chambon, 325 Nature 75, 1987) have shown that the receptors are composed of a series of conserved domains. The most highly conserved domain is the DNA binding domain located in region C (Krust et al., supra; Green and Chambon supra; and Evans and Hollenberg, 52 Cell 1, 1988) containing a 66-68 amino acid core composed of two zinc fingers (Schwabe et al., 348 Nature 458, 1990; Hard et al., 249 Science 157, 1990; and Luisi et al., 352 Nature 497, 1991) which is essential for recognition of REs. Three amino acids adjacent to the N-terminal zinc finger of the DNA binding domain, known as the P-box, are critical for DNA sequence recognition (Mader et al., 338 Nature 271, 1989; Umesomo and Evans, 57 Cell 1139, 1989; and Danielson et al., 57 Cell 1131, 1989). A subfamily composed of the glucocorticoid, mineralocorticoid, progesterone and androgen receptors contain Gly, Ser and Val at discriminatory positions of the P-box and recognize AGAACA half-sites arranged in a palindrome with a 3 bp spacer region (Mader et al., supra; Umesomo and Evans, supra; and Danielson et al., supra). The ligand binding domain, located C-terminal to the DNA binding domain in region E, is less well conserved among the receptors and contains a ligand-inducible transcriptional activation function (Green and Chambon, supra; Evans, supra; Wahli and Martinez, supra; Gronemeyer, supra; Giguere et al., 46 Cell 645, 1986; and Kumar et al., 51 Cell 941, 1987). Transcriptional activating domains have also been identified in the poorly conserved N-terminal A/B regions of the glucocorticoid and estrogen receptors (Giguere et al., supra; Kumar et al., supra; and Tora et al., 59 Cell 447, 1989).
Response elements are often found in multiple arrays, usually located upstream of the site of transcriptional initiation (Jantzen et al., supra; Martinez et al., 6 EMBO J. 3719, 1987; and Burch et al., 8 Mol. Cell. Biol. 1123, 1988). Functional analysis of two glucocorticoid response elements (GREs) located far upstream of the rat tyrosine aminotransferase (TAT) gene has shown that they combine synergistically to mediate transcriptional activation by the glucocorticoid receptor (GR) (Jantzen et al., supra). The two nonconsensus estrogen response elements (EREs) of the Xenopus vitellogenin B1 gene are virtually inactive in isolation, but together mediate estrogen-dependent transcriptional activation in transiently transfected cells (Martinez et al., supra). The degree of synergism between paired EREs or GREs is dependent on their sequence, the spacing between them and their distance from the TATA box of the promoter (Ponglikitmongkol et al., 9 EMBO J. 2221, 1990; and Schule et al., 332 Nature 87, 1988). Several studies have also shown that nuclear receptors and other classes of transcriptional regulators can combine to activate transcription synergistically (Tora et al., supra; and Schule et al., supra).
Promoters activated by specific inducible nuclear receptors are well suited for eukaryotic expression vectors since expression of genes can be regulated simply by controlling the concentration of ligand present in growth media (Kumar supra; Lee et al., 294 Nature 259, 1984; Ucker et al., 27 Cell 257, 1981; and Metzger et al., 334 Nature 31, 1988). Glucocorticoid-inducible promoters such as that of the long terminal repeat of the mouse mammary tumor virus (MMTV) have been widely used in this regard because the GR is expressed in a wide variety of mammalian cell types. The MMTV GRE is composed largely of a series of half-sites (Payvar et al., 35 Cell 381, 1983; and Scheidereit et al., 304 Nature 749, 1983). While the MMTV promoter can be induced by ligand-bound receptors for both glucocorticoids and progesterone when introduced into cells by transient transfection, it is selectively responsive to glucocorticoids when propagated on an episomal vector (Hager and Archer, in "Nuclear Hormone Receptors: Molecular Mechanisms, Cellular functions and Clinical Abnormalities," (Parker, M. G. ed.), Academic Press Ltd. 217, 1991). It is proposed that the nucleosome structure of the episomal DNA selectively inhibits access of the progesterone receptor to the response element.
Schena et al., 88 Proc. Natl. Acad. Sci. USA 10421, 1991, describe a steriod inducible expression system for plant cells in which six 26-base pair GREs are fused upstream of a plant site of transcription initiation with a plant TATA box as the sole promoter element. By providing glucocorticoid receptor and glucocorticoid hormone, inducible expression of a gene in a plant cell was achieved.